Targeted Activation of G-Protein Coupled Receptor-Mediated Ca2+ Signaling Drives Enhanced Cartilage-Like Matrix Formation.

Intracellular calcium ([Ca2+]i) signaling is a critical regulator of chondrogenesis, chondrocyte differentiation, and cartilage development. Calcium (Ca2+) signaling is known to direct processes that govern chondrocyte gene expression, protein synthesis, cytoskeletal remodeling, and cell fate. Control of chondrocyte/chondroprogenitor Ca2+ signaling has been attempted through mechanical and/or pharmacological activation of endogenous Ca2+ signaling transducers; however, such approaches can lack specificity and/or precision regarding Ca2+ activation mechanisms. Synthetic signaling platforms permitting precise and selective Ca2+ signal transduction can improve dissection of the roles that [Ca2+]i signaling plays in chondrocyte behavior. One such platform is the chemogenetic DREADD (designer receptor exclusively activated by designer drugs) hM3Dq, which activates [Ca2+]i signaling via the Gαq-PLCß-IP3-ER pathway upon clozapine N-oxide (CNO) administration. We previously demonstrated hM3Dq's ability to precisely and synthetically initiate robust [Ca2+]i transients and oscillatory [Ca2+]i signaling in chondrocyte-like ATDC5 cells. Here, we investigate the effects that long-term CNO stimulatory culture have on hM3Dq [Ca2+]i signaling dynamics, proliferation, and protein deposition in 2D ATDC5 cultures. Long-term culturing under repeated CNO stimulation modified the temporal dynamics of hM3Dq [Ca2+]i signaling, increased cell proliferation, and enhanced matrix production in a CNO dose- and frequency-dependent manner, and triggered the formation of cell condensations that developed aligned, anisotropic neotissue structures rich in cartilaginous proteoglycans and collagens, all in the absence of differentiation inducers. This study demonstrated Gαq-G-protein coupled receptor (GPCR)-mediated [Ca2+]i signaling involvement in chondroprogenitor proliferation and cartilage-like matrix production, and it established hM3Dq as a powerful tool for elucidating the role of GPCR-mediated Ca2+ signaling in chondrogenesis and chondrocyte differentiation. Impact statement Targeted activation of intracellular calcium signaling has gained attention as a cartilage tissue engineering adjuvant approach. In the present study, we demonstrated that activation of hM3Dq, an engineered chemogenetic activator of the Gαq-pathway and IP3-mediated intracellular calcium signaling, drives accelerated development of mesenchyme-like cell condensations and cartilaginous neotissue formation in chondrocyte-like cell cultures in vitro and does so without the requirement of differentiation factors/inducers. These outcomes highlight the potential of targeted/synthetic Gαq-pathway activation, specifically using novel chemogenetic approaches, to enhance the study of chondrocyte physiology and improve cartilage tissue engineering approaches.

Targeted Gq-GPCR activation drives ER-dependent calcium oscillations in chondrocytes.

The temporal dynamics of calcium signaling are critical regulators of chondrocyte homeostasis and chondrogenesis. Calcium oscillations regulate differentiation and anabolic processes in chondrocytes and their precursors. Attempts to control chondrocyte calcium signaling have been achieved through mechanical perturbations and synthetic ion channel modulators. However, such stimuli can lack both local and global specificity and precision when evoking calcium signals. Synthetic signaling platforms can more precisely and selectively activate calcium signaling, enabling improved dissection of the roles of intracellular calcium ([Ca2+]i) in chondrocyte behavior. One such platform is hM3Dq, a chemogenetic DREADD (Designer Receptors Exclusively Activated by Designer Drugs) that activates calcium signaling via the Gαq-PLCß-IP3-ER pathway upon administration of clozapine N-oxide (CNO). We previously described the first-use of hM3Dq to precisely mediate targeted, synthetic calcium signals in chondrocyte-like ATDC5 cells. Here, we generated stably expressing hM3Dq-ATDC5 cells to investigate the dynamics of Gαq-GPCR calcium signaling in depth. CNO drove robust calcium responses in a temperature- and concentration-dependent (1 pM-100 µM) manner and elicited elevated levels of oscillatory calcium signaling above 10 nM. hM3Dq-mediated calcium oscillations in ATDC5 cells were reliant on ER calcium stores for both initiation and sustenance, and the downregulation and recovery dynamics of hM3Dq after CNO stimulation align with traditionally reported GPCR recycling kinetics. This study successfully generated a stable hM3Dq cell line to precisely drive Gαq-GPCR-mediated and ER-dependent oscillatory calcium signaling in ATDC5 cells and established a novel tool to elucidate the role that GPCR-mediated calcium signaling plays in chondrocyte biology, cartilage pathology, and cartilage tissue engineering.

DREADD-based synthetic control of chondrocyte calcium signaling in vitro.

Calcium is a critical second messenger involved in chondrocyte mechanotransduction. Several distinct calcium signaling mechanisms implicated in chondrocyte mechanotransduction have been identified using mechanical perturbations or soluble signaling factors. However, these commonly used stimuli can lack specificity in the mechanisms by which they initiate calcium signaling. Synthetic tools allowing for more precise and selective regulation of calcium signaling, such as the engineered G-protein-coupled receptors known as DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), may better assist in isolating the roles of intracellular calcium ([Ca2+ ]i ) and cell activation in chondrocyte biology. One DREADD, hM3Dq, is solely activated by clozapine N-oxide (CNO) and regulates calcium activation through the Gq -PLCß-IP3 -ER pathway. Here, hM3Dq-transfected ATDC5 cells were treated with CNO (100 nM-1 µM) to establish the feasibility of using Gq -DREADDs to drive [Ca2+ ]i activation in chondrocyte-like cells. CNO administration resulted in a coordinated, dose-dependent, and transient calcium response in hM3Dq-transfected cells that resulted primarily from calcium release from the ER. Following activation via CNO administration, hM3Dq-ATDC5 cells exhibited refractory behavior and required a 4-h wash-out period to recover hM3Dq-mediated signaling. However, hM3Dq inactivation did not inhibit alternative calcium activation mechanisms in ATDC5 cells (via GSK101 or hypo-osmotic shock), nor did CNO-driven calcium signaling negatively impact ATDC5 cell health. This study established the successful use of hM3Dq for the safe, targeted, and well-controlled activation of calcium signaling in ATDC5 cells and its use as a potential tool for assessing clinically significant questions regarding calcium signaling in chondrocyte biology, cartilage pathology, and cartilage tissue engineering. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1518-1529, 2019.